HAX1-dependent control of mitochondrial proteostasis governs neutrophil granulocyte differentiation

The relevance of molecular mechanisms governing mitochondrial proteostasis to the differentiation and function of hematopoietic and immune cells is largely elusive. Through dissection of the network of proteins related to HCLS1-associated protein X-1, we defined a potentially novel functional CLPB/HAX1/(PRKD2)/HSP27 axis with critical importance for the differentiation of neutrophil granulocytes and, thus, elucidated molecular and metabolic mechanisms underlying congenital neutropenia in patients with HAX1 deficiency as well as bi- and monoallelic mutations in CLPB. As shown by stable isotope labeling by amino acids in cell culture (SILAC) proteomics, CLPB and HAX1 control the balance of mitochondrial protein synthesis and persistence crucial for proper mitochondrial function. Impaired mitochondrial protein dynamics are associated with decreased abundance of the serine-threonine kinase PRKD2 and HSP27 phosphorylated on serines 78 and 82. Cellular defects in HAX1–/– cells can be functionally reconstituted by HSP27. Thus, mitochondrial proteostasis emerges as a critical molecular and metabolic mechanism governing the differentiation and function of neutrophil granulocytes.

frequency from against public (e.g. GnomAD, ExAC and GME) and in-house databases.
Potentially causative variants were confirmed by Sanger sequencing.

Sanger sequencing
Sanger sequencing of CLPB was performed to confirm WES-detected variants and their segregation with the clinical phenotype across the family members. Genomic DNA was PCRamplified using OneTaq Polymerase (NEB), specific primers are provided in Supplementary PB210PA-1), and positively transported cells were then selected using puromycin (0.5 μg/ml).
As a control, we prepared HAX1 knockout iPS cells transfected with an empty piggybac construct with the PiggyBac transposase vector. After puromycin (0.5 μg/ml) selection, undifferentiated iPSC cells were subsequently stimulated with doxycycline (0.05 μg/ml) for 24 hours prior to immunoblotting for checking the overexpression of HAX1 and HSP27.

sgRNA design and cloning for targeted gene correction in iPS cells
SgRNA-Cas9 with fusion GFP plasmid was purchased from Addgene (PX458, cat# 48138).

FACS analysis and sorting
All antibodies used for FACS analysis are listed in the Supplemental Table 6. Upon doxycycline (0.05 μg/ml) induction, floating neutrophil-like cells were collected and shortly washed with PBS prior to staining with FACS antibodies. Mature neutrophil population was identified by gating with (Live/mcheery + /CD33 low /CD11b + ) prior to further FACS analysis.

Molecular Cloning
The coding regions of genes used in this study were amplified from homemade human cDNA and were inserted into vectors summarized in the Supplemental Table 9. PRKD2 was amplified from pDONR233-PRKD2 vector ordered from addgene. All primers have been synthetized by Eurofins Genomics and are listed in Supplement Table 5. All plasmids were sequence confirmed.

Stable transfection
In order to confer stable overexpression of indicated genes (HAX1, CLPB and HSP27),

Inhibitor studies
To inhibit the activity of PRKD family in PLB-985 cells, cells were treated with 1 μM or 3 μM CRT0066101 for 3 h at 37°C prior to analysis by SDS-PAGE and immunoblotting.

Mitochondrial isolation
PLB-985 or HeLa cells were respectively harvested and homogenized in TH buffer (300 mM

Mitochondrial carbonate extraction
Mitochondria isolated from indicated cell lines were solubilized with 0.1 M Na2CO3 at pH 10.8 or pH 11.5. As a control, mitochondria were resuspended with 1% Triton buffer on ice. After 20 min incubation on ice, mitochondrial membranes were pelleted by ultracentrifugation at 45,000 rpm, 4°C for 45 min. All samples were precipitated with trichloroacetic acid and washed with ice cold acetone before analyzed by immunoblotting.

Immunoprecipitation with Anti-FLAG M2 agarose beads
Wild-type and mutant HAX1 or CLPB Flag-tagged plasmids were transfected into semiconfluent HEK293T cells with 10 µg plasmid/10 cm dish. After 48 h, cells were pelleted and lysed in freshly prepared RIPA Buffer (450 mM NaCl, 25 mM Tris-HCl pH 7.5, 1 mM EDTA, 1% NP40, 5% Glycerol, 25 mM Na-Pyrophosphate, 50 mM Na-Fluoride, EDTA-free protease inhibitor (Roche)). The supernatant was collected and remaining debris were removed by centrifugation of 14,000 rpm, at 4°C for 15 min. Subsequently, the supernatant was incubated with anti-FLAG M2 Affinity gel (Sigma, cat# A2220) overnight at 4°C on a rotating laboratory wheel. Beads were pelleted and washed 5 times in RIPA buffer before analyzed by immunoblotting.

Immunofluorescence studies
Fixed cells were shortly washed with PBS and quenched in 50 mM NH4Cl for 30 min.
Afterwards, cells were permeabilized with 0.2% Trion for 2 min at RT prior to blocking with 0.02% Triton and 1% BSA in TBS (w/v). Coverslips were then incubated with indicated primary and secondary antibodies for 1 h at RT before DAPI staining. Nuclear staining was conducted Protein eluates were precipitated with cold acetone and resuspended in LYSE buffer (PreOmics). Bound proteins were further digested and analyzed by mass spectrometry.

Measurement of mitochondrial complexes activity
The activities of the mitochondrial complexes I and IV were assessed by the Complex I Enzyme Activity Microplate Assay Kit (abcam) and the Complex IV Human Specific Activity Microplate Assay Kit (abcam) respectively. Mitochondria adopted for the measurements were harvested on the same day.

In-gel-mass spectrometry
Mass spectrometry of Supplemental Figure. 1 was performed as previously described (2).
After immunoprecipitation of HAX1 FLAG , the eluate was subjected to SDS-PAGE and coomassie stained bands were cut out and in-gel digested. Gel pieces were processed as

SILAC labelling
Cells were cultured in RPMI 1640 media lacking two amino acids (lysine and arginine) (RPMI 1640 media for SILAC, Life technologies) with dialyzed FBS. The medium was supplemented with either "light" standard unlabelled Lysine and Arginine or "medium" isotope labelled 13 C4 --Lysine (Lys4) and 13 C6 --Arginine (Arg6) (Silantes, Munich, Germany). Cells were cultured for more than 10 passages in SILAC medium and their incorporation of "medium" amino acids was confirmed by MS analysis. For the pulse experiment with heavy SILAC amino acids, cells cultured in "medium" amino acids were washed and incubated with "heavy"-isotope labelled 13 C6 15 N2 --Lysine (Lys8) and 13 C6 15 N4 --Arginine (Arg10) (Silantes) for 24 hours (see Figure. 3A). At time 0, 6 hours and 24 hours, pulsed cells were collected and counted using cell counting chamber. 2 × 10 6 PLB-985 cells of "heavy"-pulsed cells were mixed with the same number of cells cultured in "light" medium (3). 1 × 10 6 "heavy"-pulsed+ "light" cells were washed with PBS and pelleted at 1200 rpm for 3 minutes and snap-frozen. 3 × 10 6 "heavy"-pulsed+ "light" cells were further processed for the isolation of mitochondria (see above) which were subsequently snap-frozen. Data were obtained in three independent cell clones per genotype in two entirely independent biological replicates corresponding to two rounds of SILAC labeling. Spectrophotomether (Thermo Scientifics).

Data Acquisition and Computational proteomics
Eluted peptides (0.5 μg/sample) were separated on a reverse phase 50-cm column with 75μm inner diameter, packed in-house with 1. and proteins and was determined by searching a reverse database. A maximum of two missed cleavages were allowed in the database search. Peptide identification was performed with an allowed initial precursor mass deviation up to 7 ppm and an allowed fragment mass deviation of 10 ppm. The "Match between runs" option in MaxQuant was activated.

Related to Supplemental
Proteins related to Glycolysis were selected based on Gene Ontology (GO) annotations.
Proteins with significantly different expression (*P<0.05) between WT and HAX1 -/or WT and CLPB -/are listed.  Table 4. Subunits of RCs identified in the HSP27 IP. Related to Figure 6.

Supplemental
List of subunits of RCs identified in complex with HSP27. The columns correspond to protein ID, protein name and gene name.